Brockerhoff S E, Davis T N
Department of Biochemistry, University of Washington, Seattle 98195.
J Cell Biol. 1992 Aug;118(3):619-29. doi: 10.1083/jcb.118.3.619.
Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.
利用亲和纯化的多克隆抗体，通过间接免疫荧光法将钙调蛋白定位在酿酒酵母中。钙调蛋白呈现出不对称分布，且在细胞周期中会发生变化。在未出芽的细胞中，钙调蛋白在芽出现前约10分钟集中在芽形成的假定部位。在小芽殖细胞中，钙调蛋白在整个芽中积累。随着芽的生长，钙调蛋白集中在芽尖，然后分散，最终在胞质分裂前集中在颈部区域。当野生型钙调蛋白被结合Ca2+能力受损的钙调蛋白突变体取代时，观察到相同的染色模式。因此，钙调蛋白的定位并不取决于其与Ca2+高亲和力结合的能力。用亲和纯化的抗钙调蛋白抗体和罗丹明偶联的鬼笔环肽对酵母细胞进行双重标记表明，在细胞周期中钙调蛋白和肌动蛋白集中在重叠区域。此外，使用温度敏感的钙调蛋白突变体破坏钙调蛋白功能会使肌动蛋白定位紊乱，而act1-4突变体中钙调蛋白分布随机。因此，钙调蛋白和肌动蛋白的分布相互依赖。最后，在α因子处理的细胞中，钙调蛋白定位于shmoo尖端。这种在细胞生长部位的分布表明钙调蛋白参与了酵母中的极化细胞生长。
